1. Technical Field
The present invention relates generally to wound healing assays, and more particularly, to a system and method for electrically wounding and/or monitoring a cell culture.
2. Background Art
Wound healing assays have been carried out in tissue culture for many years to monitor cell behavior, including estimating the migration and proliferative capacities of different cells and of cells under different conditions.
These assays generally involve first growing cells to form a confluent monolayer. The monolayer is then disrupted by destroying or displacing a group of cells. Common methods for disrupting the cell monolayer comprise scratching a line (i.e., manually dragging a pointed device) through the layer with any of several different tools such as a needle, razor blade, plastic pipette tip or by removing a small area of cells, e.g., with a spinning circular pad. Additionally, wounding may be carried out using a very small spinning disk to give a more reproducible area to follow. Once a wound is achieved, the wound is then microscopically observed or photographed over time to assess the rate at which the damaged area is filled in by the neighboring cells. The above-mentioned wounding methods require extensive manipulation of the cultured cells, making the wounding methods expensive and, furthermore, difficult to accurately reproduce and to verify experimental results.
As noted, after the disruption is accomplished, the area is inspected microscopically at different time intervals as the cells move in and fill the damaged area. This “healing” can take from several hours to over a day depending on the cell type, growing conditions, and the extent of the “wounded” region. The results may be presented by a series of photomicrographs; or in more sophisticated measurements, the microscopic views may be subjected to image processing such that data can be expressed in quantitative terms.
An alternative form of measuring cell behavior that replaced the commonly used microscopic observations utilizes electrical sensing. One example is disclosed in U.S. Pat. No. 5,187,096, which is hereby incorporated by reference and referred to herein as the “ECIS™ system.” Specifically, the ECIS™ system (Electric Cell-substrate Impedance Sensing) sold by Applied Biophysics, Inc., passively analyzes cell behavior by applying a weak AC current and measuring the voltage changes. The device can be used to monitor various cell behaviors, including the morphology changes and cell motions in animal cells that attach and spread out and crawl on the bottom of tissue culture vessels. In the ECIS™ system, cells are grown upon a small gold film electrode (5×10−4 cm2) mounted to the bottom of a small well; a much larger counter electrode completes the circuit using standard tissue culture medium as an electrolyte. A weak (e.g., approximately 1 microamp) AC current (usually in the frequency range from 100 to 40,000 Hz) is applied to the system. This small current results in a voltage drop across the small electrode of only a few millivolts. Voltage drops and currents this small do not affect the health of the cells.
Variations in the measured voltage comprise the measurement. As the animal cells attach and spread upon the small electrode, they force the current to flow under and between the cells resulting in changes in impedance and hence, in the measured voltage across the electrode system. These changes can be followed and provide a non-invasive means to monitor changes in cell behavior. For example, using the measured voltages, one can infer cell morphology and cell movements, which are important research measurements that form the basis of many biomedical and biological assays.
While the ECIS™ system allows for automated and passive monitoring of cell behavior following a disruption, the requirement for manipulating the cell culture to create the wound or disruption remains. In addition to requiring human intervention, the current procedure amplifies the possibility of external factors inadvertently affecting the results. Therefore, there exists a need for a wound healing method that provides data that is more quantitative and reproducible than the current methods. In addition, there exists a need for a wound healing method that requires less manipulations and thus less labor in obtaining the experimental results.